This application addresses RFA-GM-1-1009, Exceptional, Unconventional Research Enabling Knowledge Acceleration (EUREKA)(R01). The basic unit of any biological system is the cell, and malfunctions at the single-cell level can result in devastating diseases. To understand the physiology and functions of any biological system, therefore, it is important to achieve measurements with single-cell resolutions. This proposal aims to employ advanced mass spectrometric technologies which, when appropriately combined with new droplet manipulation techniques, will allow us to meet the tremendous challenge of analyzing the contents of single cells with high sensitivty and information content using mass spectrometry. Specifically, the aim of this project is: Design and construct a mass spectrometer with the appropriate interface for ionizing and analyzing the contents of single cells trapped in liquid droplets. The interface will be a droplet nanolab platform with the optical trapping and positioning of droplets in air, which will be used towards achieving the efficient ionization of single-droplet contents and ion transport for mass spectrometry analysis. Atmospheric pressure chemical ionization (APCI) will be used for the analysis of small-molecule (e.g. metabolites and lipids) contents of single cells. MALDI will be used for the analysis of peptide and protein contents of single cells. The cell is the basic unit of any biological system; to understand the functions and malfunctions of an organism will thus necessitate measurements at the single-cell level. Yet the biochemical analysis of single cells is highly challenging, owing to both the minute amounts of sample available as well as the high complexity of the sample. In this proposed project, we aim to tackle these challenges by pushing the limit of sensitivity of mass spectrometry for analyzing the minute but highly concentrated single-cell derived samples present in droplets. PUBLIC HEALTH RELEVANCE: The cell is the basic unit of any biological system; to understand the functions and malfunctions of an organism will thus necessitate measurements at the single-cell level. Yet the biochemical analysis of single cells is highly challenging, owing to both the minute amounts of sample available as well as the high complexity of the sample. In this proposed project, we aim to tackle these challenges by pushing the limit of sensitivity of mass spectrometry for analyzing the minute but highly concentrated single-cell derived samples present in droplets.